Ca²⁺ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line

1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca²⁺ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium ([Ca²⁺](i). We found that, as detected by indo-1 fluorescence, [Ca²⁺](i) increases in a slow tonic manner from 99 ± 11 nM in virgin HL-60 to 182 ± 19 nM (p<0.001) in those treated with 1,25-(OH)₂D₃ for 24 h. The first apparent rise in [Ca²⁺](i) occurs at between 6 and 12 h and parallels expression of α-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca²⁺](i) is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca²⁺](i) is associated with an increase in inositol trisphosphate-stimulated Ca²⁺ flux from intracellular stores. Interestingly, 1,25-(OH)₂D₃-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca²⁺](i) is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca²⁺](i) during 1,25-(OH)₂D₃ treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)₂D₃ is Ca²⁺-independent, expression of response to regulatory stimuli requires priming of cellular Ca²⁺ stores. The latter appears to be induced by 1,25-(OH)₂D₃ via stimulated Ca²⁺ entry through the plasma membrane.